三種不同石英晶體表面修飾法應用於腸炎弧菌之檢測

APPLYING THREE DIFFERENT SURFACE MODIFICATION METHODS FOR DETECTION OF Vibrio parahaemolyticus

林姵君(Pei-Chun Lin)

指導教授 ： 顏聰榮

大同大學/工程學院/生物工程學系所/碩士(2007年)

摘要

生物感測器是將免疫學、生物化學等使用之原理應用於創新之分析量測技術上，這種方法與傳統檢測方法具有極大之差異，其獨特性與優點，在某些分析上可以解決許多傳統分析方法不易處理的問題。本研究利用石英晶體微天秤（QCM）對微重敏感性，分別以三種不同方法〔Polyethylenimine-Glutaraldehyde（PEI-GA）、冷電漿接枝法、Protein A法〕對石英晶體表面做前處理再將抗腸炎弧菌抗體固定於QCM晶片表面製成腸炎弧菌免疫壓電晶片用以檢測腸炎弧菌，並找出最適合的表面改質方法，以期能開發出可用於臨床分析的腸炎弧菌快速檢測系統。實驗結果顯示，以冷電漿接枝法處理過的石英晶體固定抗腸炎弧菌抗體的方式效能最佳，比起PEI-GA法及Protein A法高出約1.7倍。PEI-GA法的抗腸炎弧菌抗體添加量為0.025-0.2μg，所測得的頻率變化為72-126 Hz；冷電漿接枝法的抗腸炎弧菌抗體添加量為0.0125-0.05μg，所測得的頻率變化為61.8-136 Hz；Protein A法的抗腸炎弧菌抗體添加量為0.025-0.1μg，所測得的頻率變化為72.4-112.8 Hz，此三種方法均可測得腸炎弧菌的濃度介於 CFU/mL - CFU/mL之間，由上述可得知冷電漿接枝法比PEI-GA法及Protein A法可在添加低抗腸炎弧菌抗體量即可有效偵測到腸炎弧菌，於實驗結果可得知利用冷電漿接枝法處理的QCM晶片其靈敏度及偵測效果都較PEI-GA法及Protein A法還要高。

關鍵字

石英晶體微天秤 ； 抗體固定化 ； 腸炎弧菌

並列摘要

Biosensor is a deveice for the detection of an analyte that combines a biological component with a physicochemical detector component. It consists of 3 parts：the sensitive biological element, the transducer, and the detector element. A quartz crystal microbalance (QCM) is an ultra-sensitive mass sensor. Correlation between mass and frequency is achieved by means of the sauerbrey equation. In this study, a modified antibody-coated QCM biosensor will be constructed for detcting food pathogenic bacteria Vibrio parahaemolyticus. Three immobilization methods, polyethylenimine-Glutaraldehyde（PEI-GA）, plasma grafting, and Protein A, were used to prepare the immunochips, the enteritis vibrio immunity piezoelectric crystal plate used during analysis, and to find the most suitable surface modification method. It is expected that such a system would be useful for fast routine examinations. From the results, the plasma grafting yielded the best performance, which was better than the others (the PEI-GA and the Protein) with an improvement of 1.7 times. Moreover, in the PEI-GA results there was an observable frequency shift from 72 to 126 Hz, when amounts of Vibrio parahaemolyticus O1:K1 polyclonal Ab (0.025-0.2μg) were added. There was a frequency shift in the plasma grafting from 61.8 to 136 Hz when amounts of Vibrio parahaemolyticus O1:K1 polyclonal Ab (0.0125-0.05μg) were added, and for Protein A there was a frequency shift from 72.4 to 112.8 Hz when amounts of Vibrio parahaemolyticus O1:K1 polyclonal Ab (0.025-0.1μg) were added. From the above results, we found that the concentrations of Vibrio parahaemolyticus could be detected from CFU/mL to CFU/mL by the proposed three methods. In addition, we also found that the concentrations for Vibrio parahaemolyticus could be detected by the plasma grafting in the lower concentration of Vibrio parahaemolyticus O1:K1 polyclonal Ab. Furthermore, we compared the sensitivity of these three methods. We found that the sensitivity of plasma grafting was higher than that of PEI-GA or Protein A. It can be deduced from the above results that all the above methods are capable of the fast detection of Vibrio parahaemolyticus.

並列關鍵字

Vibrio parahaemolyticus ； quartz crystal microbalance ； antibody immobilization

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